Generally, methods of producing recombinant proteins are performed using various cell lines such as E. coli, yeast and animal cells are used. These methods comprise transforming a recombinant protein expression vector into a cell line, culturing the cell line to express the target protein, and lysing the cells to purify the target protein.
These methods require a process for selecting a strain for stably expressing the recombinant protein, and then a series of processes, including cell culture, cell disruption and protein purification, should be performed. Thus, these methods require large amounts of time and effort.
In addition, cytotoxic proteins or membrane proteins are difficult to express. Thus, in the case of these proteins, many efforts are required, such as attempting various expression conditions. For this reason, there is a problem in that a time from several days to several months is required for production of a single protein.
In order to overcome this problem, there have been developed cell-free protein expression methods for expressing a protein in a reaction vessel such as a test tube within a short time without using cells, and products associated therewith. These methods are intended to express a desire protein without cell culture. These methods use a vector comprising a target gene, a cell extract obtained by isolation/purification after cell culture, and a protein expression solution including amino acids, triphosphate ribonucleic acid, and an energy source and a buffer. In these methods, a gene encoding a desired protein is transcribed into mRNA by RNA polymerase contained in a cell extract, and the mRNA is translated to the protein using the ribosome, tRNA and the like contained in the cell extract.
The cell-free protein expression method was commercialized by Roche, Promega and the like. In this method, a template DNA (e.g., expression vector, PCR product, etc.) capable of expressing protein, a cell lysate, and an expression solution including amino acid, triphosphate ribonucleic acid and an energy source, are added to a test tube, and then allowed to react at a suitable temperature (30 to 40° C.) for a suitable time (1-3 hours) to express the target protein. This method is advantageous over a method of expressing a protein using cells, in that a significantly reduced amount of time is required, and cytotoxic proteins or membrane proteins can be expressed.
In addition, systems capable of producing proteins in a convenient manner by automatically performing cell-free protein expression were developed.
Such systems include RTS (Rapid Translation System) developed by Roche. The RTS can perform cell-free protein expression with high efficiency. In this system, a dialysis membrane is disposed in the bottom of a reaction vessel in order to prevent a reaction from being stopped due to energy source exhaustion and the accumulation of a protein synthesis inhibitor such as phosphoric acid during the progression of cell-free protein expression. For dialysis, this reaction vessel is placed in an at least 10-fold volume of a vessel containing a small-molecule expression solution in order to maximize the reaction. As described in Kim, D.-M. and Choi, C.-Y., Biotechnol. Prog., 12(5):645, 1996, this principle is based on the principle according to which the expression level of a protein can be increased up to 10 times by continuously replacing a small-molecule expression solution containing triphosphate ribonucleic acid, an amino acid and an energy source through a dialysis membrane.
However, this method has a problem in that, a small-molecule expression solution having a volume corresponding to at least 10 times the volume of an expression solution should also be used, the overall size of the reaction vessel becomes greater, and thus the size of the system significantly increases when various kinds of proteins are to be simultaneously synthesized in parallel. In addition, the system is not automated, and can perform only a function of stirring a reaction solution at a certain temperature. Thus, a separate process such as the purification of a synthesized protein after the reaction should be performed.
The RIKEN institute (Japan) automated protein expression by constructing this large-scale system, although the detailed structure of the system was not explained (Alexander Spirin & James R. Swartz, Cell-Free Protein Synthesis, p 103, FIG. 6.3B). Yaeta Endo et al. developed a cell-free method of expressing a protein using a wheat germ extract, and developed an automated system based on a principle different from the RTS system. Also, Yaeta Endo et al. could increase the protein expression yield by diluting a cell-free protein expression solution with a small-molecule expression solution and concentrating the dilution (US 2006/0257997 A1).
However, the above automated system have problems in that it is expensive due to its complex structure, and has no protein purification function, like the RTS system, and it is difficult to produce several kinds of proteins.
Meanwhile, a sample containing a recombinant protein expressed by a cell culture or cell-free protein expression method contains various kinds of proteins in addition to a target protein. To easily purify the target protein from this sample, a technology of attaching an affinity tag (histidine tag) is used. In this technology, when a histidine tag encoding sequence is introduced into a template gene, the histidine tag is attached to the C or N terminus of a protein produced from the template gene, and the target protein can be easily obtained in high purity by isolating/purifying the protein using the affinity between the histidine tag and metal ions (nickel ions, cobalt ions, etc.).
However, the affinity tag that is used for purification can adversely affect the activity and structure of the target protein. For this reason, for the production of target proteins for protein function research and medical purposes, a process of removing the tag is required.
The present inventors (Bioneer) developed the automated protein synthesis system ExiProgen™ (Korean Patent Laid-Open Publication Nos. 10-2011-0121588 and 10-2013-0023091) and a related kit. This system is configured to automatically perform a cell-free protein expression reaction and purify an expressed protein. In this system, a template DNA and solutions required for a reaction can be automatically mixed in order to automatically perform a protein expression reaction, and can be maintained at a suitable temperature for the reaction. Also, all processes, including the synthesis of RNA from DNA and the expression of a protein from the RNA, can be maintained at the optimum temperature. Further, in order to easily purify a target protein from a mixture sample containing the expressed protein, an affinity tag such as oligohistidine is attached to the target protein, and in this state, the target protein is expressed, and is automatically purified using a magnetic bead having divalent metal ions (nickel ions, cobalt ions, etc.) attached thereto. The above ExiProgen™ and kit can simultaneously express and purify various kinds of proteins, but have problems in that, because protein expression is performed in a batch fashion, an energy source is exhausted with the passage of time, and is terminated by the accumulation of a protein expression inhibitor, and thus the level of a finally expressed protein is low.
In order to increase the expression level of a protein, a small-molecule expression solution containing triphosphate ribonucleic acid, an amino acid and an energy source should be continuously supplied, and protein expression inhibitors should be removed. In order to automatically perform such operations, new types of reactor and system are required.
The present inventors have made efforts to develop a system and a kit, which can overcome the shortcoming of low protein expression efficiency of the automated protein synthesizer ExiProgen™, and at the same time, increase the expression level of a target protein while eliminating the need to increase the size of the system due to the use of a small-molecule expression solution in an amount 10 times or more the amount of an expression solution. As a result, the present inventors have developed a system capable of increasing the expression level of a target protein by continuously supplying a small-molecule expression solution through a dialysis membrane during a cell-free protein expression reaction and removing protein expression inhibitors.
It was found that the system developed by the present inventors has the effect of increasing the efficiency of protein expression by automatically supplying a small amount of a small-molecule expression solution in several steps, can automatically purify an expressed protein, like the conventional system ExiProgen™, and can automatically replace a target protein solution, which contains a high-concentration eluate, with a desired protein storage buffer after purification.
In addition, the present inventors have found that, when a protein from which an affinity tag used for purification is to be removed is produced, a pure target protein can be produced by automatically removing the affinity tag by treatment with a protease, which recognizes and cleaves a specific amino acid sequence of the protein, during purification of the protein, thereby completing the present invention.